In order to optimize the immune response to malaria synthetic peptide vaccines, parasite-derived "universal" T cell epitopes, i.e. epitopes that can bind to the majority of class II molecules, will be identified in malaria preerythrocytic stage antigens and the specificity and effector functions elicited by vaccines containing these epitomes determined. In the current grant, we propose to study universal T cell epitopes that we have identified using two experimental methods. The first method was direct, or empirical, and utilized malaria-specific human T cell clones to identify a potential universal T cell epitope in aa 326-345 of the P. falciparum (NF54) CS protein. The epitope defined by this method was shown to bind to multiple DR and DQ molecules in vitro and elicit antibody responses in mice of diverse genetic backgrounds. The second method we have used identities "theoretical" universal T cell epitopes by analyzing protein sequences for regions that contain multiples high affinity HLA peptide binding motifs. Since submission of our original application, we have obtained preliminary results demonstrating the ability to use motif analysis to identify three potential universal T cell epitopes in the P. falciparum TRAP/SSP2 protein. The molecular basis of promiscuous binding to HLA class II molecules of the empirically-defined and the predicted P. falciparum universal T cell epitopes will be defined using in vitro peptide binding assays. Synthetic peptides containing the malaria universal T cell epitopes will be tested for recognition by cells of sporozoite-immunized volunteers and murine strains of different haplotypes. New methods for the construction of complex multimeric peptide containing multiple parasite- derived T and B cell epitopes, synthesized by MAP technology or polyoxime peptide chemistry, will also be explored. Tri-epitope MAP or polyoxime constructs, containing the P. falciparum CS or Trap universal T cell eptiopes, incombination with the CS repeat B cell epitope, will be tested for their ability to induce malaria-specific antibody and cell-mediated immune responses in murine strains of diverse genetic backgrounds. The muring malaria analogues of CS and TRAP universal epitopes inprotectin against sporozoite challenge. The correlation for peptide/MHC interactions in vitro with function immunogenicity in vivo will validate the use of approaches for the development of malaria vaccines capable of eliciting protective levels of humoral and cellular immunity in individuals of diverse genetic backgrounds.